Treatment and diagnosis of cancer

ABSTRACT

The present invention is directed to the use of antibodies or binding portions thereof, probes, ligands, or other biological agents which either recognize an extracellular domain of prostate specific membrane antigen or bind to and are internalized with prostate specific membrane antigen. These biological agents can be labeled and used for detection of cancerous tissues, particularly cancerous tissues proximate to or containing vascular endothelial cells, which express an extracellular domain of prostate specific membrane antigen. The labeled biological agents can also be used to detect normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof. They also can be used alone or bound to a substance effective to ablate or kill such cells as a therapy for prostate or other cancers. Also disclosed are four hybridoma cell lines, each of which produces a monoclonal antibody recognizing extracellular domains of prostate specific membrane antigens of normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof.

The present application claims the benefit of U.S. Provisional PatentApplication 60/022,125, filed Jul. 18, 1996, and is acontinuation-in-part of U.S. patent application Ser. No. 08/838,682,filed Apr. 9, 1997, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/016,976, filed May 6, 1996.

FIELD OF THE INVENTION

The present invention relates to the treatment and diagnosis of cancerwith biological agents.

BACKGROUND OF THE INVENTION

In spite of improved treatments for certain forms of cancer, it is stilla leading cause of death in the United States. Since the chance forcomplete remission of cancer is, in most cases, greatly enhanced byearly diagnosis, it is very desirable that physicians be able to detectcancers before a substantial tumor develops. However, the development ofmethods that permit rapid and accurate detection of many forms ofcancers continues to challenge the medial community. One suchillustrative form of cancer is prostate cancer.

Prostate cancer is the most common cancer in men with an estimated317,000 cases in 1996 in the United States. It is the second leadingcause of death among men who die from neoplasia with an estimated 40,000deaths per year. Prompt detection and treatment is needed to limitmortality caused by prostate cancer.

Detection of Prostate Cancer

When it metastasizes, prostatic dancer has a distinct predilection forbone and lymph nodes. Saitoh et al., “Metastatic Patterns of ProstaticCancer. Correlation Between Sites And Number Of Organs Involved,”Cancer, 54:3078-3084 (1984). At the time of clinical diagnosis, as manyas 25% of patients have bone metastasis demonstrable by radionuclidescans. Murphy, G. P., et al., “The National Survey Of Prostate Cancer InThe United States By The American College Of Surgeons,” J. Urol.,127:928-939 (1982). Accurate clinical evaluation of nodal involvementhas proven to be difficult. Imaging techniques such as computedtomography (“CT”) or magnetic resonance (“MR”) imaging are unable todistinguish metastatic prostate cancer involvement of lymph nodes bycriterion other than size (i.e., >1 cm). Therefore, by definition, theseimaging modalities are inherently insensitive in the detection of smallvolume (<1 cm) disease as well as non-specific in the detection oflarger volume adenopathy. A recent study assessed the accuracy of MR inpatients with clinically localized prostate cancer. Rifkin et al.,“Comparison Of Magnetic Resonance Imaging And Ultrasonography In StagingEarly Prostate Cancer,” N. Engel. J. Med., 323:621-626 (1990). In thisstudy, 194 patients underwent an MR and 185 of these patients had alymph node dissection. 23 (13%) patients had pathologically involvedlymph nodes. MR was suspicious in only 1 of these 23 cases resulting ina sensitivity of 4%. Similar results have also been noted with CT scans.Gasser et al., “MRI And Ultrasonography In Staging Prostate Cancer,” N.Engl. J. Med. (Correspondence), 324(7):49-495 (1991).

The elevation of serum acid phosphatase activity in patients havingmetastasized prostate carcinoma was first reported by Gutman et al., J.Clin. Invest 17:473 (1938). In cancer of the prostate, prostatic acidphosphatase is released from the cancer tissue into the blood streamwith the result that the total serum acid phosphatase level can begreatly increased above normal values. Numerous studies of this enzymeand its relation to prostatic cancer have been made since that time,e.g. Yam, Amer. J. Med. 56:604 (1974). However, the measurement of serumacid phosphatase is elevated in about 65-90 percent of patients havingcarcinoma of the prostate with bone metastasis; in about 30 percent ofpatients without roentgenological evidence of bone metastasis; and inabout only 5-10 percent of patients lacking clinically demonstrablemetastasis.

Prior art attempts to develop a specific test for prostatic acidphosphatase have met with only limited success, because techniques whichrely on enzyme activity on a so-called “specific” substrate cannot takeinto account other biochemical and immunochemical differences among themany acid phosphatases which are unrelated to enzyme activity ofprostate origin. In the case of isoenzymes, i.e. genetically definedenzymes having the same characteristic enzyme activity and a similarmolecular structure but differing in amino acid sequences and/or contentand, therefore, immunochemically distinguishable, it would appearinherently impossible to distinguish different isoenzyme forms merely bythe choice of a particular substrate. It is, therefore, not surprisingthat none of these prior art methods is highly specific for the directdetermination of prostatic acid phosphatase activity; e.g. see Cancer5:236 (1952); J. Lab. Clin. Med. 82:486 (1973); Clin. Chem. Acta. 44:21(1973); and J. Physiol. Chem. 356:1775 (1975).

In addition to the aforementioned problems of non-specificity whichappear to be inherent in many of the prior art reagents employed for thedetection of prostate acid phosphatase, there have been reports ofelevated serum acid phosphatase associated with other diseases, whichfurther complicates the problem of obtaining an accurate clinicaldiagnosis of prostatic cancer. For example, Tuchman et al., Am. J. Med.27:959 (1959) noted that serum acid phosphatase levels appear to beelevated in patients with Gaucher's disease.

Due to the inherent difficulties in developing a “specific” substratefor prostate acid phosphatase, several researchers have developedimmunochemical methods for the detection of prostate acid phosphatase.However, the previously reported immunochemical methods have drawbacksof their own which have precluded their widespread acceptance. Forexample, Shulman et al., Immunology 93:474 (1964) described animmuno-diffusion test for the detection of human prostate acidphosphatase. Using antisera prepared from a prostatic fluid antigenobtained by rectal massage from patients with prostatic disease, nocross-reactivity precipitin line was observed in the double diffusiontechnique against extracts of normal kidney, testicle, liver, and lung.However, this method has the disadvantages of limited sensitivity, evenwith the large amounts of antigen employed, and of employing antiserawhich may cross-react with other, antigenically unrelated serum proteincomponents present in prostatic fluid.

WO 79/00475 to Chu et. al. describes a method for the detection ofprostatic acid phosphatase isoenzyme patterns associated with prostaticcancer which obviates many of the above drawbacks. However, practicalproblems are posed by the need for a source of cancerous prostate tissuefrom which the diagnostically relevant prostatic acid phosphataseisoenzyme patterns associated with prostatic cancer are extracted forthe preparation of antibodies thereto.

In recent years, considerable effort has been spent to identify enzymeor antigen markers for various types of malignancies with the viewtowards developing specific diagnostic reagents. The ideal tumor markerwould exhibit, among other characteristics, tissue or cell-typespecificity. Previous investigators have demonstrated the occurrence ofhuman prostate tissue-specific antigens.

Treatment of Prostate Cancer

As described in W. J. Catalona, “Management of Cancer of the Prostate,”New Engl. J. Med., 331(15):996-1004 (1994), the management of prostatecancer can be achieved by watchful waiting, curative treatment, andpalliation.

For men with a life expectancy of less than 10 years, watchful waitingis appropriate where low-grade, low-stage prostate cancer is discoveredat the time of a partial prostatectomy for benign hyperplasia. Suchcancers rarely progress during the first five years after detection. Onthe other hand, for younger men, curative treatment is often moreappropriate.

Where prostate cancer is localized and the patient's life expectancy is10 years or more, radical prostatectomy offers the best chance foreradication of the disease. Historically, the drawback of this procedureis that most cancers had spread beyond the bounds of the operation bythe time they were detected. However, the use of prostate-specificantigen testing has permitted early detection of prostate cancer. As aresult, surgery is less extensive with fewer complications. Patientswith bulky, high-grade tumors are less likely to be successfully treatedby radical prostatectomy.

After surgery, if there are detectable serum prostate-specific antigenconcentrations, persistent cancer is indicated. In many cases,prostate-specific antigen concentrations can be reduced by radiationtreatment. However, this concentration often increases again within twoyears.

Radiation therapy has also been widely used as an alternative to radicalprostatectomy. Patients generally treated by radiation therapy are thosewho are older and less healthy and those with higher-grade, moreclinically advanced tumors. Particularly preferred procedures areexternal-beam therapy which involves three dimensional, conformalradiation therapy where the field of radiation is designed to conform tothe volume of tissue treated; interstitial-radiation therapy where seedsof radioactive compounds are implanted using ultrasound guidance; and acombination of external-beam therapy and interstitial-radiation therapy.

For treatment of patients with locally advanced disease, hormonaltherapy before or following radical prostatectomy or radiation therapyhas been utilized. Hormonal therapy is the main form of treating menwith disseminated prostate cancer. Orchiectomy reduces serumtestosterone concentrations, while estrogen treatment is similarlybeneficial. Diethylstilbestrol from estrogen is another useful hormonaltherapy which has a disadvantage of causing cardiovascular toxicity.When gonadotropin-releasing hormone agonists are administeredtestosterone concentrations are ultimately reduced. Flutamide and othernonsteroidal, anti-androgen agents block binding of testosterone to itsintracellular receptors. As a result, it blocks the effect oftestosterone, increasing serum testosterone concentrations and allowspatients to remain potent—a significant problem after radicalprostatectomy and radiation treatments.

Cytotoxic chemotherapy is largely ineffective in treating prostatecancer. Its toxicity makes such therapy unsuitable for elderly patients.In addition, prostate cancer is relatively resistant to cytotoxicagents.

Use of Monoclonal Antibodies in Prostate Cancer Detection and Treatment

Theoretically, radiolabeled monoclonal antibodies (“mAbs”) offer thepotential to enhance both the sensitivity and specificity of detectingprostatic cancer within lymph nodes and elsewhere. While many mAbs havepreviously been prepared against prostate related antigens, none ofthese mAbs were specifically generated with an imaging objective inmind. Nevertheless, the clinical need has led to evaluation of some ofthese mAbs as possible imaging agents. Vihko et al., “Radioimaging ofProstatic Carcinoma With Prostatic Acid Phosphatase-SpecificAntibodies,” Biotechnology in Diagnostics, 131-134 (1985); Babaian etal., “Radioimmunological Imaging of Metastatic Prostatic Cancer With111-Indium-Labeled Monoclonal Antibody PAY 276,” J. Urol., 137:439-443(1987); Leroy et al., “Radioimmunodetection Of Lymph Node Invasion InProstatic Cancer. The Use Of Iodine 123 (123-I)-Labeled MonoclonalAnti-Prostatic Acid Phosphatase (PAP) 227 A F (ab′) 2 Antibody FragmentsIn Vivo,” Cancer, 64:1-5 (1989); Meyers et al., “Development OfMonoclonal Antibody Imaging Of Metastatic Prostatic Carcinoma,” TheProstate, 14:209-220 (1989).

In some cases, the monoclonal antibodies developed for detection and/ortreatment of prostate cancer recognize antigens specific to malignantprostatic tissues. Such antibodies are thus used to distinguishmalignant prostatic tissue (for treatment or detection) from benignprostatic tissue. See U.S. Pat. No. 4,970,299 to Bazinet et al. and U.S.Pat. No. 4,902,615 to Freeman et al.

Other monoclonal antibodies react with surface antigens on all prostateepithelial cells whether cancerous or benign. See U.S. Pat. No.4,446,122 and Re 33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwanet al., and U.S. Pat. No. 5,055,404 to Ueda et al. However, the antigensdetected by these monoclonal antibodies are present in the blood and,therefore, compete with antigens at tumor sites for the monoclonalantibodies. This causes background noise which makes the use of suchantibodies inadequate for in vivo imaging. In therapy, such antibodies,if bound to a cytotoxic agent, could be harmful to other organs.

Horoszewicz et al., “Monoclonal Antibodies to a New Antigenic Marker inEpithelial Prostatic Cells and Serum of Prostatic Cancer Patients,”Anticancer Research, 7:927-936 (1987) (“Horoszewicz”) and U.S. Pat. No.5,162,504 to Horoszewicz describe an antibody, designated 7E11, whichrecognizes prostate specific membrane antigen (“PSMA”). Israeli et al.,“Molecular Cloning of a Complementary DNA Encoding a Prostate-specificMembrane Antigen,” Cancer Research, 53:227-230 (1993) (“Israeli”)describes the cloning and sequencing of PSMA and reports that PSMA isprostate-specific and shows increased expression levels in metastaticsites and in hormone-refractory states. Other studies have indicatedthat PSMA is more strongly expressed in prostate cancer cells relativeto cells from the normal prostate or from a prostate with benignhyperplasia. Furthermore, PSMA is not found in serum (Troyer et al.,“Detection and Characterization of the Prostate-Specific MembraneAntigen (PSMA) in Tissue Extracts and Body Fluids,” Int. J. Cancer,62:552-558 (1995)).

These characteristics make PSMA an attractive target for antibodymediated targeting for imaging and therapy of prostate cancer. Imagingstudies using indium-labeled 7E11 have indicated that the antibodylocalizes quite well to both the prostate and to sites of metastasis. Inaddition, 7E11 appears to have clearly improved sensitivity fordetecting lesions compared to other currently available imagingtechniques, such as CT and MR imaging or bone scan. Bander, “CurrentStatus of Monoclonal Antibodies for Imaging and Therapy of ProstateCancer,” Sem. In Oncology, 21:607-612 (1994).

However, the use of 7E11 and other known antibodies to PSMA to mediateimaging and therapy has several disadvantages. First, PSMA is anintegral membrane protein known to have a short intracellular tail and along extracellular domain. Biochemical characterization and mapping(Troyer et al., “Biochemical Characterization and Mapping of the7E11-C5.3 Epitope of the Prostate-specific Membrane Antigen,” Urol.Oncol., 1:29-37 (1995)) have shown that the epitope or antigenic site towhich the 7E11 antibody binds is present on the intracellular portion ofthe molecule. Because antibody molecules do not, under normalcircumstances, cross the cell membrane unless they bind to theextracellular portion of a molecule and become translocatedintracellularly, the 7E11 antibody does not have access to its antigenictarget site in an otherwise healthy, viable cell.

Consequently, imaging using 7E11 is limited to the detection of deadcells within tumor deposits. Additionally, the therapeutic use of the7E11 antibody is limited, because only cells that are already dead ortissue containing a large proportion of dead cells can be effectivelytargeted.

Although the inadequacies and problems in the diagnosis and treatment ofone particular type of cancer are the focus of the preceding discussion,prostate cancer is merely a representative model. The diagnosis andtreatment of numerous other cancers have similar problems.

The present invention is directed to overcoming the deficiencies ofprior art antibodies in diagnosing and treating prostate and other typesof cancer.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of ablating orkilling cancerous cells. The process involves providing a biologicalagent which, when contacted with an extracellular domain of prostatespecific membrane antigen, recognizes the extracellular domain ofprostate specific membrane antigen. These biological agents arecontacted with vascular endothelial cells proximate to the cancerouscells under conditions effective to permit both binding of thebiological agent to the vascular endothelial cells proximate to thecancerous cells and killing or ablating of the cancerous cells. Thebiological agent can be used alone or can be bound to a substanceeffective to kill or ablate the cancerous cells upon binding of thebiological agent to vascular endothelial cells that are proximate to thecancerous cells.

In a particularly preferred embodiment of the method of ablating orkilling cancerous cells in accordance with the present invention, thebiological agent, when contacted with an extracellular domain ofprostate specific membrane antigen, binds to and is internalized withthe prostate specific membrane antigen of such cells. Preferredbiological agents for use in the method of ablating or killing cancerouscells in accordance with the present invention are antibodies or bindingportions thereof, probes, or ligands. The methods of the presentinvention are particularly useful in killing or ablating renal,urothelial, colon, rectal, lung, and breast cancerous cells andcancerous cells of metastatic adenocarcinoma to the liver.

Another aspect of the present invention relates to a method of detectingcancerous tissue in a biological sample. This method involves providinga biological agent which, when contacted with an extracellular domain ofprostate specific membrane antigen, binds to the extracellular domain ofprostate specific membrane antigen. The biological agent is bound to alabel effective to permit detection of vascular endothelial cellsproximate to or within the cancerous tissue upon binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue. The biological sample is contacted with thebiological agent having a label under conditions effective to permitbinding of the biological agent to the vascular endothelial cellsproximate to or within the cancerous tissue in the biological sample.The presence of cancerous tissue in the biological sample is detected bydetection of the label.

In a particularly preferred embodiment of the method of detectingcancerous tissue in accordance with the present invention, thebiological agent is one that, when contacted with an extracellulardomain of prostate specific membrane antigen, binds to and isinternalized with the prostate specific membrane antigen. Preferredbiological agents for use in the method of detecting cancerous tissue inaccordance with the present invention are antibodies or binding portionsthereof, probes, or ligands. The method is especially useful indetecting renal, urothelial, colon, rectal, lung, and breast canceroustissue and cancerous tissue of metastatic adenocarcinoma to the liver.

Still another aspect of the present invention relates to a method ofablating or killing normal, benign hyperplastic, and cancerous prostateepithelial cells. The process involves providing a biological agentwhich recognizes an extracellular domain of prostate specific membraneantigen. The biological agent can be used alone or can be bound to asubstance effective to kill the cells upon binding of the biologicalagent to the cells. These biological agents are then contacted with thecells under conditions effective to permit both binding of thebiological agent to the extracellular domain of the prostate specificmembrane antigen and killing or ablating of the cells.

In a particularly preferred embodiment of the method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells in accordance with the present invention, the biological agentbinds to and is internalized with the prostate specific membrane antigenof such cells. Preferred biological agents for use in the method ofablating or killing normal, benign hyperplastic, and cancerous prostateepithelial cells in accordance with the present invention are antibodiesor binding portions thereof, probes, or ligands.

Another aspect of the present invention relates to a method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells orportions thereof in a biological sample. This method involves providinga biological agent which binds to an extracellular domain of prostatespecific membrane antigen. The biological agent is bound to a labeleffective to permit detection of the cells or portions thereof uponbinding of the biological agent to the cells or portions thereof. Thebiological sample is contacted with the biological agent having a labelunder conditions effective to permit binding of the biological agent tothe extracellular domain of the prostate specific membrane antigen ofany of the cells or portions thereof in the biological sample. Thepresence of any cells or portions thereof in the biological sample isdetected by detection of the label.

In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent binds to andis internalized with the prostate specific membrane antigen of suchcells. Preferred biological agents for use in the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention are antibodies or binding portionsthereof, probes, or ligands.

Another aspect of the present invention pertains to a biological agentthat recognizes an extracellular domain of prostate specific membraneantigen. In a preferred embodiment, the isolated biological agent bindsto and is internalized with the prostate specific membrane antigen.Preferred isolated biological agents which recognize an extracellulardomain of prostate specific membrane antigen in accordance with thepresent invention are isolated antibodies or binding portions thereof,probes, or ligands. Hybridoma cell lines that produce monoclonalantibodies of these types are also disclosed.

The biological agents of the present invention recognize theextracellular domain of antigens of normal, benign hyperplastic, andcancerous prostate epithelial cells. Unlike the 7E11 antibody, whichrecognizes an epitope of prostate-associated antigens which are exposedextracellularly only after cell lysis, the biological agents of thepresent invention bind to antigenic epitopes which are extracellularlyexposed in living prostate cells. Using the biological agents of thepresent invention, living, unfixed normal, benign hyperplastic, andcancerous prostate epithelial cells can be targeted, which makestreatment and diagnosis more effective. In a preferred embodiment fortreating prostate cancer, the biological agents of the present inventionalso bind to and are internalized with the prostate specific membraneantigen, which permits the therapeutic use of intracellularly actingcytotoxic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 on the surface of LNCaP cells after incubation at 4° C.

FIG. 2 is an immuno-electron micrograph of LNCaP cells treated withgold-labeled monoclonal antibody J591 after 5 minutes incubation at 37°C.

FIG. 3 is an immuno-electron micrograph of LNCaP cells treated withgold-labeled monoclonal antibody J591 after 10 minutes incubation at 37°C.

FIG. 4 is an immuno-electron micrograph of LNCaP cells treated withgold-labeled monoclonal antibody J591 after 15 minutes incubation at 37°C.

FIG. 5 is an immuno-electron micrograph of LNCaP cells treated withgold-labeled monoclonal antibody J591 after 15 minutes at 37° C. showingJ591 within endosomes.

FIG. 6 summarizes the sequencing strategy of the heavy chain ofmonoclonal antibody J591.

FIG. 7 shows the nucleotide sequence of the heavy chain of monoclonalantibody J591 (designated SEQ. ID. No. 1), the nucleotide sequence ofthe corresponding reverse, non-coding strand (designated SEQ. ID. No.2), and the corresponding deduced amino acid sequences (designated SEQ.ID. Nos. 3, 4, and 5).

FIG. 8 is a comparison of the heavy chain of monoclonal antibody J591with the consensus sequence for Mouse Heavy Chains Subgroup IIA.

FIG. 9 summarizes the sequencing strategy of the kappa light chain ofmonoclonal antibody J591.

FIG. 10 shows the nucleotide sequences of the kappa light chain ofmonoclonal antibody J591 (designated SEQ. ID. No. 9), the nucleotidesequence of the corresponding reverse, non-coding strand (designatedSEQ. ID. No. 10), and the corresponding deduced amino acid sequence(designated SEQ. ID. Nos. 11, 12, and 13).

FIG. 11 is a comparison of the kappa light chain of monoclonal antibodyJ591 with the consensus sequence for Mouse Kappa Chains Subgroup V.

FIGS. 12A-12F are micrographs (250× magnification) showing theimmunohistochemical reactivity of mAb J591 to neovasculature of variouscarcinomas.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells. The process involves providing a biological agent, such as anantibody or binding portion thereof, probe, or ligand, which binds to anextracellular domain of prostate specific membrane antigen of (i.e., aportion of prostate specific membrane antigen which is external to) suchcells. The biological agent can be used alone or can be bound to asubstance effective to kill the cells upon binding of the biologicalagent to the cells. These biological agents are then contacted with thecells under conditions effective to permit both binding of thebiological agent to the extracellular domain of the prostate specificmembrane antigen and killing or ablating of the cells. In its preferredform, such contacting is carried out in a living mammal by administeringthe biological agent to the mammal under conditions effective to permitboth binding of the biological agent to the extracellular domain of theprostate specific membrane antigen and killing or ablating of the cells.Such administration can be carried out orally or parenterally.

In a particularly preferred embodiment of the method of ablating orkilling normal, benign hyperplastic, and cancerous prostate epithelialcells in accordance with the present invention, the biological agentbinds to and is internalized with the prostate specific membrane antigenof such cells. Again, the biological agent can be used alone.Alternatively, the biological agent can be bound to a substanceeffective to kill the cells upon binding of the biological agent toprostate specific membrane antigen and upon internalization of thebiological agent with the prostate specific membrane antigen.

The mechanism by which the biological agent is internalized with theprostate specific membrane antigen is not critical to the practice ofthe present invention. For example, the biological agent can induceinternalization of the prostate specific membrane antigen.Alternatively, internalization of the biological agent can be the resultof routine internalization of prostate specific membrane antigen.

The above-described biological agents. (i.e., biological agents, such asan antibody or binding portion thereof, probe, or ligand which, whencontacted with an extracellular domain of prostate specific membraneantigen, recognizes the extracellular domain of prostate specificmembrane antigen and, preferably, is internalized therewith) can be usedto ablate or kill cancerous cells. In this aspect of the presentinvention, the biological agent can be used alone or can be bound to asubstance effective to kill the cancerous cells upon binding of thebiological agent to vascular endothelial cells proximate thereto. Thesebiological agents are contacted with vascular endothelial cellsproximate to the cancerous cells. The contacting is carried out underconditions that are effective to permit binding of the biological agentto the vascular endothelial cells proximate to the cancerous cells and,in addition, that are effective to kill or ablate the cancerous cells.The mechanism by which the cancerous cells are killed or ablated is notcritical to the practice of the present invention. For example, thecancerous cells can be killed or ablated directly by the biologicalagent as a consequence of their proximity to the vascular endothelialcells to which the biological agent binds. Alternatively, the biologicalagent can kill, ablate, or otherwise change the properties of thevascular endothelial cells to which it binds so that blood flow to thecancerous cells proximate thereto is stopped or otherwise reduced,thereby causing the cancerous cells to be killed or ablated. Thus, themethod of the present invention is particularly useful for killing orablating vascular endothelial cells in cancerous tissue as well as thecancerous cells contained in cancerous tissue.

In a particularly preferred embodiment of the method of ablating orkilling cancerous cells in accordance with the present invention, thebiological agent employed is one that, when contacted with anextracellular domain of prostate specific membrane antigen, binds to andis internalized with the extracellular domain of prostate specificmembrane antigen. The methods of the present invention are particularlyuseful to kill or ablate cancerous prostate epithelial cells as well ascancerous cells other than cancerous prostate epithelial cells. Examplesof cancerous cells which are not cancerous prostate epithelial cells arerenal, urothelial, colon, rectal, lung, and breast cancerous cells andcancerous cells of metastatic adenocarcinoma to the liver. Although themethod of the present invention can be used to kill or ablate any cellwhich expresses an extracellular domain of prostate specific membraneantigen or a portion thereof or whose subsistence is dependent uponcells which express an extracellular domain of prostate specificmembrane antigen or a portion thereof, the method of the presentinvention is particularly useful to kill or ablate cancerous cells,because the vascular endothelial cells supplying blood to canceroustissues (e.g., tumors, collections of cancerous cells, or othercancerous masses) express an extracellular domain of prostate specificmembrane antigen, irrespective of the type of cancer involved. Incontrast, vascular endothelial cells supplying blood to normal tissuesdo not express an extracellular domain of prostate specific membraneantigen.

Another aspect of the present invention relates to a method of detectingnormal, benign hyperplastic, and cancerous epithelial cells or portionsthereof in a biological sample. This method involves providing abiological agent, such as an antibody or binding portion thereof, probe,or ligand, which binds to an extracellular domain of prostate specificmembrane antigen of such cells. The biological agent is bound to a labeleffective to permit detection of the cells or portions (e.g., prostatespecific membrane antigen or fragments thereof liberated from suchnormal, benign hyperplastic, and cancerous cells) thereof upon bindingof the biological agent to the cells or portions thereof. The biologicalsample is contacted with the biological agent having a label underconditions effective to permit binding of the biological agent to theextracellular domain of the prostate specific membrane antigen of any ofthe cells or portions thereof in the biological sample. The presence ofany cells or portions thereof in the biological sample is detected bydetection of the label. In its preferred form, such contacting iscarried out in a living mammal and involves administering the biologicalagent to the mammal under conditions effective to permit binding of thebiological agent to the prostate specific membrane antigen of any of thecells or portions thereof in the biological sample. Again, suchadministration can be carried out orally or parenterally.

The method of the present invention can be used to screen patients fordiseases associated with the presence of normal, benign hyperplastic,and cancerous epithelial cells or portions thereof. Alternatively, itcan be used to identify the recurrence of such diseases, particularlywhen the disease is localized in a particular biological material of thepatient. For example, recurrence of prostatic disease in the prostaticfossa may be encountered following radical prostatectomy. Using themethod of the present invention, this recurrence can be detected byadministering a short range radiolabeled antibody to the mammal and thendetecting the label rectally, such as with a transrectal detector probe.

Alternatively, the contacting step can be carried out in a sample ofserum or urine or other body fluids, such as to detect the presence ofPSMA in the body fluid. When the contacting is carried out in a serum orurine sample, it is preferred that the biological agent recognizesubstantially no antigens circulating in the blood other than PSMA.Since intact prostate cells do not excrete or secrete PSMA into theextracellular environment, detecting PSMA in serum, urine, or other bodyfluids generally indicates that prostate cells are being lysed. Thus,the biological agents and methods of the present invention can be usedto determine the effectiveness of a prostate cancer treatment protocolby monitoring the level of PSMA in serum, urine or other body fluids.

In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent, such as theantibody or binding portion thereof, probe, or ligand, binds to and isinternalized with the prostate specific membrane antigen of such cells.Again, the biological agent is bound to a label effective to permitdetection of the cells or portions thereof upon binding of thebiological agent to and internalization of the biological agent with theprostate specific membrane antigen.

Another aspect of the present invention relates to a method of detectingcancerous tissue in a biological sample. This method involves providingthe above-described biological agent (i.e., a biological agent, such asan antibody or binding portion thereof, probe, or ligand which, whencontacted with an extracellular domain of prostate specific membraneantigen, recognizes the extracellular domain of prostate specificmembrane antigen). The biological agent is bound to a label that iseffective to permit detection of vascular endothelial cells proximate toor within the cancerous tissue upon binding of the biological agent tovascular endothelial cells proximate to or within the cancerous tissue.The biological sample is then contacted with the biological agent havinga label. Contacting is carried out under conditions effective to permitbinding of the biological agent to the vascular endothelial cellsproximate to or within the cancerous tissue in the biological sample.The presence of cancerous cells or portions thereof in the biologicalsample is detected by detection of the label.

Rather than contacting the entire biological sample with the biologicalagent, it is contemplated that a portion of the biological sample can beused. For example, a tissue biopsy sample can be contacted with thebiological agent to determine the presence of cancerous tissue in thetissue biopsy sample as well as in the larger biological sample fromwhich it is taken. Alternatively, the biological agent can be contactedwith a serum or urine sample to acertain whether any vascularendothelial cells expressing an extracellular domain of prostatespecific membrane antigen are present therein. Since vascularendothelial cells expressing an extracellular domain of prostatespecific membrane antigen are found in the vasculature of canceroustissues but not in the vasculature of normal tissues, detection of thelabel in a serum or urine sample indicates the presence of canceroustissue in the larger biological sample from which it is taken (e.g., apatient).

In a particularly preferred embodiment of the method of detectingcancerous tissues in accordance with the present invention, thebiological agent employed is one that, when contacted with anextracellular domain of prostate specific membrane antigen, binds to andis internalized with the prostate specific membrane antigen. The methodsof the present invention can be used to detect cancerous prostateepithelial cells as well as cancerous tissues containing cancerous cellsother than cancerous prostate epithelial cells. Examples of canceroustissues containing cancerous cells other than cancerous prostateepithelial cells which can be detected with the methods of the presentinvention include renal, urothelial, colon, rectal, lung, and breastcancerous tissue and cancerous tissue of metastatic adenocarcinoma tothe liver.

As indicated above, biological agents suitable for either killing,ablating, or detecting cancerous cells and normal, benign hyperplastic,and cancerous prostate epithelial cells include antibodies, such asmonoclonal or polyclonal antibodies. In addition, antibody fragments,half-antibodies, hybrid derivatives, probes, and other molecularconstructs may be utilized. These biological agents, such as antibodies,binding portions thereof, probes, or ligands, bind to extracellulardomains of prostate specific membrane antigens or portions thereof innormal, benign hyperplastic, and cancerous prostate epithelial cells. Asa result, when practicing the methods of the present invention to kill,ablate, or detect normal, benign hyperplastic, and cancerous prostateepithelial cells, the biological agents bind to all such cells, not onlyto cells which are fixed or cells whose intracellular antigenic domainsare otherwise exposed to the extracellular environment. Consequently,binding of the biological agents is concentrated in areas where thereare prostate epithelial cells, irrespective of whether these cells arefixed or unfixed, viable or necrotic. Additionally or alternatively,these biological agents, such as antibodies, binding portions thereof,probes, or ligands, bind to and are internalized with prostate specificmembrane antigens or portions thereof in normal, benign hyperplastic,and cancerous prostate epithelial cells.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The resulting fused cells, orhybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned, and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature 256:495 (1975), which is hereby incorporated byreference.

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the protein or polypeptide of the presentinvention. Such immunizations are repeated as necessary at intervals ofup to several weeks to obtain a sufficient titer of antibodies.Following the last antigen boost, the animals are sacrificed and spleencells removed.

Fusion with mammalian myeloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511(1976), which is hereby incorporated by reference). This immortal cellline, which is preferably murine, but may also be derived from cells ofother mammalian species, including but not limited to rats and humans,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and to have goodfusion capability. Many such cell lines are known to those skilled inthe art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbit's which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain syntheticsurfactant adjuvant pluronic polyols, or pulverized acrylamide gelcontaining the protein or polypeptide after. SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. Ultimately, the rabbits are euthenized with pentobarbital 150mg/Kg IV. This and other procedures for raising polyclonal antibodiesare disclosed in E. Harlow, et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, the processes of the presentinvention encompass use of binding portions of such antibodies. Suchbinding portions include Fab fragments, F(ab′)₂ fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118(N.Y. Academic Press 1983), which is hereby incorporated by reference.

Alternatively, the processes of the present invention can utilize probesor ligands found either in nature or prepared synthetically byrecombinant DNA procedures or other biological or molecular procedures.Suitable probes or ligands are molecules which bind to the extracellulardomains of prostate specific membrane antigens identified by themonoclonal antibodies of the present invention. Other suitable probes orligands are molecules which bind to and are internalized with prostatespecific membrane antigens. Such probes or ligands can be, for example,proteins, peptides, lectins, or nucleic acid probes.

It is particularly preferred to use the monoclonal antibodies identifiedbelow in Table 1. TABLE 1 ATCC Designation for Monoclonal Antibody NameHybridoma Cell Line E99 HB-12101 J415 HB-12109 J533 HB-12127 J591HB-12126These antibodies can be used alone or as a component in a mixture withother antibodies or other biological agents to treat cancers or imagecancerous tissues (particularly the vascular endothelial cells therein)or prostate epithelial cells with varying surface antigencharacteristics.

Regardless of whether the biological agents are used for treatment ordiagnosis, they can be administered orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes, such as, that of the nose, throat, and bronchialtubes. They may be administered alone or with pharmaceutically orphysiologically acceptable carriers, excipients, or stabilizers, and canbe in solid or liquid form such as, tablets, capsules, powders,solutions, suspensions, or emulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing thebiological agent, such as an antibody or binding portion thereof, of thepresent invention and a carrier, for example, lubricants and inertfillers such as, lactose, sucrose, or cornstarch. In another embodiment,these compounds are tableted with conventional tablet bases such aslactose, sucrose, or cornstarch in combination with binders like acacia,cornstarch, or gelatin, disintegrating agents such as, cornstarch,potato starch, or alginic acid, and a lubricant like stearic acid ormagnesium stearate.

The biological agent of the present invention may also be administeredin injectable dosages by solution or suspension of these materials in aphysiologically acceptable diluent with a pharmaceutical carrier. Suchcarriers include sterile liquids such as water and oils, with or withoutthe addition of a surfactant and other pharmaceutically andphysiologically acceptable carrier, including adjuvants, excipients orstabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions.

For use as aerosols, the biological agent of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The materials of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

The biological agents may be utilized to detect cancerous tissues(particularly the vascular endothelial cells therein) and normal, benignhyperplastic, and cancerous prostate epithelial cells in vivo. This isachieved by labeling the biological agent, administering the labeledbiological agent to a mammal, and then imaging the mammal.

Examples of labels useful for diagnostic imaging in accordance with thepresent invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹mTc, ³²P,¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein andrhodamine, nuclear magnetic resonance active labels, positron emittingisotopes detectable by a positron emission tomography (“PET”) scanner,chemiluminescers such as luciferin, and enzymatic markers such asperoxidase or phosphatase. Short-range radiation emitters, such asisotopes detectable by short-range detector probes, such as atransrectal probe, can also be employed. These isotopes and transrectaldetector probes, when used in combination, are especially useful indetecting prostatic fossa recurrences and pelvic nodal disease. Thebiological agent can be labeled with such reagents using techniquesknown in the art. For example, see Wensel and Meares, Radioimmunoimagingand Radioimmunotherapy, Elsevier, N.Y. (1983), which is herebyincorporated by reference, for techniques relating to the radiolabelingof antibodies. See also, D. Colcher et al., “Use of MonoclonalAntibodies as Radiopharmaceuticals for the Localization of HumanCarcinoma Xenografts in Athymic Mice”, Meth. Enzymol. 121: 802-816(1986), which is hereby incorporated by reference.

A radiolabeled biological agent of this invention can be used for invitro diagnostic tests. The specific activity of a tagged biologicalagent, such as a tagged antibody, binding portion thereof, probe, orligand, depends upon the half-life, the isotopic purity of theradioactive label, and how the label is incorporated into the biologicalagent. Table 2 lists several commonly-used isotopes, their specificactivities and half-lives. In immunoassay tests, the higher the specificactivity, in general, the better the sensitivity. TABLE 2 SpecificActivity of Pure Isotope Isotope (Curies/mole) Half-Life ¹⁴C 6.25 × 10¹5720 years ³H 2.01 × 10⁴ 12.5 years ³⁵S 1.50 × 10⁶ 87 days ¹²⁵I 2.18 ×10⁶ 60 days ³²P 3.16 × 10⁶ 14.3 days ¹³¹I 1.62 × 10⁷ 8.1 days

Procedures for labeling biological agents with the radioactive isotopeslisted in Table 2 are generally known in the art. Tritium labelingprocedures are described in U.S. Pat. No. 4,302,438, which is herebyincorporated by reference. Iodinating, tritium labeling, and ³⁵Slabeling procedures especially adapted for murine monoclonal antibodiesare described by Goding, J. W. (supra, pp 124-126) and the referencescited therein, which are hereby incorporated by reference. Otherprocedures for iodinating biological agents, such as antibodies, bindingportions thereof, probes, or ligands, are described by Hunter andGreenwood, Nature 144:945 (1962), David et al., Biochemistry13:1014-1021 (1974) and U.S. Pat. Nos. 3,867,517 and 4,376,110, whichare hereby incorporated by reference. Radiolabeling elements which areuseful in imaging include ¹²³I, ¹³¹I, ¹¹¹In, and ^(99m)Tc, for example.Procedures for iodinating biological agents are described by Greenwood,F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J., Biochem. J.113:299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297(1971), which are hereby incorporated by reference. Procedures for^(99m)Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. etal. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,New York: Masson 111-123 (1982) and the references cited therein, whichare hereby incorporated by reference. Procedures suitable for¹¹¹In-labeling biological agents are described by Hnatowich, D. J. etal., J. Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J.Applied Radiation, 35:554-557 (1984), and Buckley, R. G. et al.,F.E.B.S. 166:202-204 (1984), which are hereby incorporated by reference.

In the case of a radiolabeled biological agent, the biological agent isadministered to the patient, is localized to the tumor bearing theantigen with which the biological agent reacts, and is detected or“imaged” in vivo using known techniques such as radionuclear scanningusing e.g., a gamma camera or emission tomography. See e.g., A. R.Bradwell et al., “Developments in Antibody Imaging”, MonoclonalAntibodies for Cancer Detection and Therapy, R. W. Baldwin et al.,(eds.), pp. 65-85 (Academic Press 1985), which is hereby incorporated byreference. Alternatively, a positron emission transaxial tomographyscanner, such as designated Pet VI located at Brookhaven NationalLaboratory, can be used where the radiolabel emits positrons (e.g., ¹¹C,¹⁸F, ¹⁵O, and ¹³N).

Fluorophore and chromophore labeled biological agents can be preparedfrom standard moieties known in the art. Since antibodies and otherproteins absorb light having wavelengths up to about 310 nm, thefluorescent moieties should be selected to have substantial absorptionat wavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluorescers and chromophores are described by Stryer, Science,162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry,41:843-868 (1972), which are hereby incorporated by reference. Thebiological agents can be labeled with fluorescent chromophore groups byconventional procedures such as those disclosed in U.S. Pat. Nos.3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated byreference.

One group of fluorescers having a number of the desirable propertiesdescribed above are the xanthene dyes, which include the fluoresceinsderived from 3,6-dihydroxy-9-henylxanthhydrol and resamines andrhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

Biological agents can be labeled with fluorchromes or chromophores bythe procedures described by Goding, J. (supra, pp 208-249). Thebiological agents can be labeled with an indicating group containing theNMR-active ¹⁹F atom, or a plurality of such atoms inasmuch as (i)substantially all of naturally abundant fluorine atoms are the ¹⁹Fisotope and, thus, substantially all fluorine-containing compounds areNMR-active; (ii) many chemically active polyfluorinated compounds suchas trifluoracetic anhydride are commercially available at relatively lowcost, and (iii) many fluorinated compounds have been found medicallyacceptable for use in humans such as the perfluorinated polyethersutilized to carry oxygen as hemoglobin replacements. After permittingsuch time for incubation, a whole body NMR determination is carried outusing an apparatus such as one of those described by Pykett, ScientificAmerican, 246:78-88 (1982), which is hereby incorporated by reference,to locate and image cancerous tissues (particularly the vascularendothelial cells therein) and prostate epithelial cells.

In cases where it is important to distinguish between regions containinglive and dead prostate epithelial cells or to distinguish between liveand dead prostate epithelial cells, the antibodies of the presentinvention (or other biological agents of the present invention), labeledas described above, can be coadministered along with an antibody orother biological agent which recognizes only living or only deadprostate epithelial cells labeled with a label which can bedistinguished from the label used to label the subject antibody. Bymonitoring the concentration of the two labels at various locations ortimes, spatial and temporal concentration variations of living and deadnormal, benign hyperplastic, and cancerous prostate epithelial cells canbe ascertained. In particular, this method can be carried out using thelabeled antibodies of the present invention, which recognize both livingand dead epithelial prostate cells, and labeled 7E11 antibodies, whichrecognize only dead epithelial prostate cells.

The biological agents can also be utilized to kill or ablate cancerouscells and normal, benign hyperplastic, and cancerous prostate epithelialcells in vivo. This involves using the biological agents by themselvesor with a cytotoxic drug to which the biological agents of the presentinvention (i.e., biological agents recognizing normal, benignhyperplastic, and cancerous prostate epithelial cells) are bound. Thisinvolves administering the biological agents bonded to a cytotoxic drugto a mammal requiring such treatment. In the case of normal,benign-hyperplastic, and cancerous prostate epithelial cells, since thebiological agents recognize prostate epithelial cells, any such cells towhich the biological agents bind are destroyed. Although suchadministration may destroy normal prostate epithelial cells, this is notproblematic, because the prostate is not required for life or survival.Although the prostate may indirectly contribute to fertility, this isnot likely to be a practical consideration in patients receiving thetreatment of the present invention. In the case of cancerous tissues,since the biological agents recognize vascular endothelial cells thatare proximate to cancerous cells, binding of the biologicalagent/cytotoxic drug complex to these vascular endothelial cellsdestroys them, thereby cutting off the blood flow to the proximatecancerous cells and, thus, killing or ablating these cancerous cells.Alternatively, the biological agents, by virtue of their binding tovascular endothelial cells that are proximate to cancerous cells, arelocalized proximate to the cancerous cells. Thus, by use of suitablebiological agents (including those containing substances effective tokill cells nondiscriminatingly but only over a short range), cells incancerous tissue (including cancerous cells) can be selectively killedor ablated.

The biological agents of the present invention may be used to deliver avariety of cytotoxic drugs including therapeutic drugs, a compoundemitting radiation, molecules of plants, fungal, or bacterial origin,biological proteins, and mixtures thereof. The cytotoxic drugs can beintracellularly acting cytotoxic drugs, such as short-range radiationemitters, including, for example, short-range, high-energy α-emitters.

Enzymatically active toxins and fragments thereof are exemplified bydiphtheria toxin A fragment, nonbinding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins,certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII andPAP-S), Morodica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin,and enomycin, for example. Procedures for preparing enzymatically activepolypeptides of the immunotoxins are described in WO84/03508 andWO85/03508, which are hereby incorporated by reference. Certaincytotoxic moieties are derived from adriamycin, chlorambucil,daunomycin, methotrexate, neocarzinostatin, and platinum, for example.

Procedures for conjugating the biological agents with the cytotoxicagents have been previously described. Procedures for conjugatingchlorambucil with antibodies are described by Flechner, I, EuropeanJournal of Cancer, 9:741-745 (1973); Ghose, T. et al., British MedicalJournal, 3:495-499 (1972); and Szekerke, M., et al., Neoplasma,19:211-215 (1972), which are hereby incorporated by reference.Procedures for conjugating daunomycin and adriamycin to antibodies aredescribed by Hurwitz, E. et al., Cancer Research, 35:1175-1181 (1975)and Arnon, R. et al. Cancer Surveys, 1:429-449 (1982), which are herebyincorporated by reference. Procedures for preparing antibody-ricinconjugates are described in U.S. Pat. No. 4,414,148 and by Osawa, T., etal. Cancer Surveys, 1:373-388 (1982) and the references cited therein,which are hereby incorporated by reference. Coupling procedures as alsodescribed in EP 86309516.2, which is hereby incorporated by reference.

In a particularly preferred embodiment of the present invention,especially well-suited for killing or ablating normal, benignhyperplastic, and cancerous prostate epithelial cells, a firstbiological agent is conjugated with a prodrug which is activated onlywhen in close proximity with a prodrug activator. The prodrug activatoris conjugated with a second biological agent according to the presentinvention, preferably one which binds to a non-competing site on theprostate specific membrane antigen molecule. Whether two biologicalagents bind to competing or non-competing binding sites can bedetermined by conventional competitive binding assays. For example,monoclonal antibodies J591, J533, and E99 bind to competing bindingsites on the prostate specific membrane antigen molecule. Monoclonalantibody J415, on the other hand, binds to a binding site which isnon-competing with the site to which J591, J533, and E99 bind. Thus, forexample, the first biological agent can be one of J591, J533, and E99,and the second biological agent can be J415. Alternatively, the firstbiological agent can be J415, and the second biological agent can be oneof J591, J533, and E99. Drug-prodrug pairs suitable for use in thepractice of the present invention are described in Blakely et al.,“ZD2767, an Improved System for Antibody-directed Enzyme Prodrug TherapyThat Results in Tumor Regressions in Colorectal Tumor Xenografts,”Cancer Research, 56:3287-3292 (1996), which is hereby incorporated byreference.

Alternatively, the biological agent can be coupled to high energyradiation emitters, for example, a radioisotope, such as ¹³¹I, aγ-emitter, which, when localized at the tumor site, results in a killingof several cell diameters. See, e.g., S. E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985),which is hereby incorporated by reference. Other suitable radioisotopesinclude α-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters,such as ¹⁸⁶Re and ⁹⁰Y. Radiotherapy is expected to be particularlyeffective, because prostate epithelial cells and vascular endothelialcells within cancers are relatively radiosensitive.

Where the biological agents are used alone to kill or ablate cancerouscells or prostate epithelial cells, such killing or ablation can beeffected by initiating endogenous host immune functions, such ascomplement-mediated or antibody-dependent cellular cytotoxicity.

The biological agent of the present invention can be used and soldtogether with equipment, as a kit, to detect the particular label.

Biological agents of the present invention can be used in conjunctionwith other therapeutic treatment modalities. Such other treatmentsinclude surgery, radiation, cryosurgery, thermotherapy, hormonetreatment, chemotherapy, vaccines, and other immunotherapies.

Also encompassed by the present invention is a method of killing orablating which involves using the biological agents for prophylaxis. Forexample, these materials can be used to prevent or delay development orprogression of prostate or other cancers.

Use of the therapeutic methods of the present invention to treatprostate and other cancers has a number of benefits. Since thebiological agents according to the present invention only targetcancerous cells (such as cells of cancerous tissues containing vascularendothelial cells) and prostate epithelial cells, other tissue isspared. As a result, treatment with such biological agents is safer,particularly for elderly patients. Treatment according to the presentinvention is expected to be particularly effective, because it directshigh levels of biological agents, such as antibodies or binding portionsthereof, probes, or ligands, to the bone marrow and lymph nodes whereprostate cancer metastases and metastases of many other cancerspredominate. Moreover, the methods of the present invention areparticularly well-suited for treating prostate cancer, because tumorsites for prostate cancer tend to be small in size and, therefore,easily destroyed by cytotoxic agents. Treatment in accordance with thepresent invention can be effectively monitored with clinical parameters,such as, in the case of prostate cancer, serum prostate specific antigenand/or pathological features of a patient's cancer, including stage,Gleason score, extracapsular, seminal, vesicle or perineural invasion,positive margins, involved lymph nodes, etc. Alternatively, theseparameters can be used to indicate when such treatment should beemployed.

Because the biological agents of the present invention bind to livingprostate cells, therapeutic methods for treating prostate cancer usingthese biological agents are much more effective than those which targetlysed prostate cells. For the same reasons, diagnostic and imagingmethods which determine the location of living normal, benignhyperplastic, or cancerous prostate epithelial cells (as well asvascular endothelial cells within cancers) are much improved byemploying the biological agents of the present invention. In addition,the ability to differentiate between living and dead prostate cells canbe advantageous, especially to monitor the effectiveness of a particulartreatment regimen.

Hybridomas E99, J415, J533, and J591 have been deposited pursuant to,and in satisfaction of, the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with the American Type Culture Collection(“A.T.C.C.”) at 12301 Parklawn Drive, Rockville, Md. 20852. HybridomaE99 was deposited on May 2, 1996, and received A.T.C.C. DesignationNumber HB-12101. Hybridoma J415 was deposited on May 30, 1996, andreceived A.T.C.C. Designation Number HB-12109. Hybridomas J533 and J591were deposited on Jun. 6, 1996, and received A.T.C.C. DesignationNumbers HB-12127 and HB-12126, respectively.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 Human Tissues

Fresh specimens of benign and malignant tissues were obtained from theDepartment of Pathology of New York Hospital Cornell University MedicalCenter (“NYH-CUMC”),

Example 2 Tissue Culture

Cultured cell lines of human cancers were obtained from the Laboratoryof Urological Oncology of NYH-CUMC. The prostate cancer cell lines PC-3(Mickey, D. D., et al., “Characterization Of A Human ProstateAdenocarcinoma Cell Line (DU145) As A Monolayer Culture And As A SolidTumor In Athymic Mice,” Prog. Clin. Biol. Res., 37:67-84 (1980), whichis hereby incorporated by reference), DU-145 (Mickey, D. D., et al.,“Characterization Of A Human Prostate Adenocarcinoma Cell Line (DU145)As A Monolayer Culture And As A Solid Tumor In Athymic Mice,” Prog.Clin. Biol. Res., 37:67-84 (1980), which is hereby incorporated byreference), and LNCaP (Horoszewicz, J. S., et al., “LNCaP Model Of HumanProstatic Carcinoma,” Cancer Res., 43:1809-1818 (1983), which is herebyincorporated by reference) were obtained from the American Type CultureCollection (Rockville, Md.). Hybridomas were initially cloned inRPMI-1640 medium supplemented with 10% FCS, 0.1 mM nonessential aminoacids, 2 mM L-glutamine, 100 units/ml of penicillin, 100 ug/ml ofstreptomycin and HAT medium (GIBCO, Grand Island, N.Y.). Subclones werecultured in the same medium without aminopterin.

Example 3 Preparation of Mouse Monoclonal Antibodies

Female BALB/c mice were immunized intraperitoneally with LNCaP (6×10⁶cells) three times at 2 week intervals. A final intraperitoneal boosterimmunization was administered with fresh prostate epithelial cells whichhad been grown in vitro. Three days later, spleen cells were fused withSP-2 mouse myeloma cells utilizing standard techniques (Ueda, R., etal., “Cell Surface Antigens Of Human Renal Cancer Defined By MouseMonoclonal Antibodies: Identification Of Tissue-Specific KidneyGlycoproteins,” Proc. Natl. Acad. Sci. USA, 78:5122-5126 (1981), whichis hereby incorporated by reference). Supernatants of the resultingclones were screened by rosette and complement cytotoxicity assaysagainst viable LNCaP. Clones which were positive by these assays werescreened by immunochemistry vs normal kidney, colon, and prostate.Clones which were LNCap⁺/NmlKid⁻/colon⁻/prostate⁺ were selected andsubcloned 3 times by limiting dilution. The immunoglobulin class ofcultured supernatant from each clone was determined by immunodiffusionusing specified rabbit antisera (Calbiochem, San Diego, Calif.). mAbswere purified using the MAPS-II kit (Bio-Rad, Richmond, Calif.).

Example 4 Biotinylation of mAbs

Purified mAbs were dialyzed in 0.1 M NaHCO₃ for 2 hours. One ml of mAbat 1 mg/ml was mixed with 0.1 ml of biotinamidocaproateN-hydroxysuccinamide ester (Sigma) in dimethylsulfoxide (1 mg/ml) andstirred for 4 hours at room temperature. Unbound biotin was removed bydialysis against phosphate buffered saline (“PBS”).

Example 5 Immunohistochemical Staining of Prostate Tissues

Cryostat sections of prostate tissues were placed inside rings of Falcon3034 plate covers (Becton-Dickenson, Lincoln Park, N.J.) previouslycoated with 0.45% gelatin solution as described in Marusich, M. F., “ARapid Method For Processing Very Large Numbers Of Tissue Sections ForImmunohistochemical Hybridoma Screening,” J. Immunol. Methods,111:143-145 (1988), which is hereby incorporated by reference. Plateswere stored at −80° C. Cryostat sections were fixed with 2%paraformaldehyde in PBS for 10 min at room temperature, and, afterwashing with PBS, endogenous peroxidase activity was blocked bytreatment with 0.3% hydrogen peroxide in PBS for 10 min at roomtemperature. After sections were incubated with 2% BSA in PBS for 20min, mAbs were added for 60 min at room temperature. Slides wereextensively washed with PBS and incubated with peroxidase-conjugatedrabbit anti-mouse Ig (DAKO Corp., Santa Barbara, Calif.) diluted 1:100in. 10% normal human serum in PBS for 60 min at room temperature. Aftera diaminobenzidine reaction, sections were counterstained withhematoxylin.

Example 6 Serological Analysis

The anti-mouse immunoglobulin mixed hemadsorption assay was performed asdescribed in Ueda, R., et al., “Cell Surface Antigens Of Human RenalCancer Defined By Mouse Monoclonal Antibodies: Identification OfTissue-Specific Kidney Glycoproteins,” Proc. Natl. Acad. Sci. USA,78:5122-5126 (1981), which is hereby incorporated by reference. Toprepare the indicator cells, anti-mouse Ig (DAKO Corp.) was conjugatedto type O human RBC using 0.01% chromium chloride. Serological assayswere performed on cells previously plated in Terasaki plates (Nunc,Denmark). Antibodies were incubated with target cells at roomtemperature for 1 hour. Target cells were then washed, and indicatorcells added for 1 hour.

Example 7 Immunoprecipitation

LNCaP cells (2×10⁷) were biotinylated with biotin-NHSS (at finalconcentration of 5 mM) for 30 minutes on ice. After washing, thebiotinylated cells were resuspended in 1 ml lysis buffer (20 mM Tris/HClpH 8.0, 1 mM EDTA, 1 mM PMSF, 1% triton X-100) for 30 min on ice. Thesuspension was centrifuged at 1500 g×100 min at 4° C., and thesupernatant was centrifuged at 12,000 rpm×15 min at 4° C. The resultinglysate was preabsorbed with rabbit or goat anti-mouse IgG-coatedpansorbin for 1 hour at 4° C. The pre-absorbed lysate was incubated withthe mAb overnight at 4° C. Rabbit or goat anti-mouse Ig-coated agarosebeads were added for 2 hours at 4° C. and then washed. The beads wereresuspended in Tris-base/NaCl, added to sample buffer with2-mercaptoethanol, and boiled for 5 min. After centrifuging, thesupernatant was run on an SDS-PAGE 12% gel. The gel was transferred to anitrocellulose membrane which was blocked and stained withstraptavidin-peroxidase. The membrane was developed withdiaminobenzidine (“DAB”).

Sequential immunoprecipitation was similar except that the lysate wasinitially pre-cleared with one mAb overnight at 4° C. A second mAb wasthen used to immunoprecipitate the pre-cleared lysate.

Approximately 2000 clones were screened, of which four clones wereselected as described in Example 3, above. After subcloning,supernatants from the 4 hybridomas, E99, J415, J533, and J591, wereassayed by immunofluorescence against viable (i.e. unfixed) LNCaP,immunoprecipitation, and sequential immunoprecipitation to confirmreactivity to PSMA.

The immunofluorescence study using the LNCaP target cell (describedoriginally in Horoszewicz, which is hereby incorporated by reference, tomake the 7E11 antibody and the prototype cell line for expression forPSMA) shows that E99 antibody binds to and renders viable LNCaP cellsimmunofluorescent. This is in contrast to the 7E11 antibody, which, asnoted originally in Horoszewicz, which is hereby incorporated byreference, gives only poor or no binding to viable LNCaP cells butexhibits strong binding once the cells are fixed (killed).

The reactivities of the four mAbs with normal human tissues wereexamined immunohistochemically; these results are presented in Table 3.TABLE 3 Reactivity of mAbs with human normal tissues by indirectimmunoperosidase staining E99 J415 J533 J591 Tissues (γ₃) (γ₁) (γ₁) (γ₁)Prostate* ● ● ● ● Kidney Glomerulus ◯ ◯ ◯ ◯ Prox. Tubule ▪ ▪ ▪ ▪ Ureter◯ ◯ ◯ ◯ Bladder ◯ ◯ ◯ ◯ Testis ◯ ◯ ◯ ◯ Uterus ◯ Esophagus ◯ ◯ ◯ ◯ SmallIntestine ◯ ◯ ◯ ◯ Stomach ◯ ◯ ◯ ◯ Colon ◯ ◯ ◯ ◯ Spleen ◯ ◯ ◯ ◯ Thyroid ◯◯ ◯ ◯ Lung ◯ ◯ ◯ ◯ Pancreas ◯ ◯ ◯ ◯ Liver ◯ ◯ ◯ ◯ BPH 0-3⁺ 0-3⁺ 0-4⁺0-4⁺ Prostate Cancer 0-3⁺ 0-3⁺ 0-4⁺ 0-4⁺ LNCaP (scid) 3⁺ 3⁺ 4⁺ 4⁺ LuCaP(scid) 0-2⁺ 0-2⁺ 0-3⁺ 0-3⁺● - positive; ▪ - weak, heterogeneous; ◯ - negativeThe above sequential immunoprecipitaion study showed that 7E11, E99,J415, J533, and J591 bind to the same molecule, i.e. PSMA.

Example 8 Western Blot Analysis

To confirm that antibodies E99, J415, J533, and J591 precipitate anidentical band to the 7E11 antibody (i.e., PSMA), Western Blot analyseswere performed. Seminal plasma (400 μg/lane) or LNCaP lysate were loadedinto lanes of 12% SDS-PAGE gels. After electrophoresis, the gels aretransferred to nitrocellulose membranes. The membranes were blocked with5% dry milk/Tris-buffered saline-tween 20 (“TBST”) for 60 min at roomtemperature. After washing, the membranes were incubated with primarymAb for 60 min at room temperature. After repeat washing, the membraneswere incubated with sheep anti-mouse-Ig-peroxidase 1/5000 in 5% drymilk/TBST for 60 min at room temperature. After repeat washing, themembranes were developed using a chemiluminescent tag designated “ECL”(Amersham Life Sciences, International, Arlington Heights, Ill.)according to the manufacturer's directions. The results of the WesternBlot experiment are presented in Table 4. TABLE 4 Western blot dataSample 7E11 E99 J415 J533 J591 Prostatic 100 KD 100 KD 100 KD 100 KD 100KD (seminal) band band band band band fluid LNCaP 100 KD & 100 KD & 100KD & 100 KD & 100 KD & cell lysate 200 KD 200 KD 200 KD 200 KD 200 KDbands bands bands bands bands

Example 9 mAb Reactivity to External Domain of PSMA

To confirm cell surface (external) expression of the detected PSMA,fresh, viable LNCaP cells were tested, without fixation, in vitro, byimmunofluorescence. LNCaP cells were washed and incubated with mAb for 1hour at room temperature and then with a rabbit anti-mouseIg-fluorescein (DAKO Corp., Santa Barbara, Calif.). Wells were read witha fluorescent microscope. Negative control consisted of anisotype-matched irrelevant mAb, while an anti-class I MHC mAb served asa positive control.

Immunofluorescence and rosette assay results are presented in Table 5.TABLE 5 Comparison of 7E11 with new mAbs LNCaP viable cells 7E11 E99J415 J533 J591 Immunoflu- neg 3+ 3+ 4+ 4+ orescence Rosette neg + + + +assay LNCaP-fixed +++ ++++ +++ ++ +++

Example 10 Competition Studies

A competition study was carried out to determine whether J591, J533,E99, and J415 detected the same or different antigenic sites (epitopes)of the prostate specific membrane antigen molecule using the followingprocedure.

Plates were coated with LNCaP cell line lysate as a source of prostatespecific membrane antigen and washed to remove unbound material. “Cold”(unlabeled) monoclonal antibody was incubated on the plate for 1 hour atroom temperature to allow binding to its antigenic site. Subsequently, asecond monoclonal antibody, labeled either with biotin or ¹²⁵I, wasadded for an additional hour. Plates were washed to remove unboundmaterial. The amount of the second monoclonal antibody bound to theprostate specific membrane antigen-coated plate was determined either byavidin-alkaline phosphatase in an enzyme-linked immunoassay (in the caseof biotin-labeled second monoclonal antibody) or by physically countingthe well in a gamma counter (in the case of ¹²⁵I-labeled secondmonoclonal antibody). Controls consisted of using the same monoclonalantibody both cold and labeled to define “100% competition” or usingmonoclonal antibody to a totally different molecule (e.g., monoclonalantibody I-56, which detects inhibin, a prostate related proteindifferent from prostate specific membrane antigen) to define “0%competition”.

The results indicated that J591, J533, and E99 each interfere, compete,or block binding of one another but do not block binding of J415 andvice versa. 7E11/CYT356, known to bind PSMA at a different(intracellular) site, did not block any of J591, J533, E99, or J415.

Having pairs of monoclonal antibodies which bind to non-competing sitespermits development of antibody sandwich assays for detecting solubleantigens, such as solubilized prostate specific membrane antigen orfragment thereof, in, for example, body fluids. For example, the antigen(e.g., prostate specific membrane antigen or a fragment thereof) couldbe “captured” from body fluid with J591 and, in another step, detectedby labeled J415.

In another setting, e.g. treatment, one could increase antibody bindingby using a combination of non-competing monoclonal antibodies. Forexample, assuming the non-competing sites are each represented once onthe prostate specific membrane antigen molecule, adding a combination ofJ591 plus J415 would bind twice as many monoclonal antibody molecules aseither monoclonal antibody alone. Binding two non-competing antigenicbinding sites also can result in greater antigen cross-linking and,perhaps, increased internalization. Furthermore, since the two detectedsites are physically located on the same prostate specific membraneantigen molecule, the binding of two monoclonal antibody molecules tothat single prostate specific membrane antigen molecule puts the twomonoclonal antibody molecules in close proximity to each other, asetting which provides optimal drug-prodrug interaction. For example,monoclonal antibody J591 can be conjugated with an inactive pro-drug andJ415 can be conjugated with a pro-drug activator. Since prodrug andactivator would be bound in close proximity only at the site of prostatespecific membrane antigen-expressing cells (e.g., prostate cancercells), prodrug activation to the active form would occur only at thosesites.

Example 11 Microscopy

Confocal microscopy and immuno-electron microscopy demonstrated thatE99, J591, J533, and J415 are bound to the cell membrane atclathrin-coated pits and then rapidly internalize into endosomes(cytoplasmic vesicles). FIGS. 1-4 are immuno-electron micrographs whichfollow the interaction of gold-labeled monoclonal antibody J591 with thecell surface as a function of time. In these figures, the location ofthe monoclonal antibody is indicated by the black dots.

Viable LNCaP cells were incubated with J591 for one hour at 4° C. Thecells were washed and then held at 37° C. for 0, 5, 10, or 15 minutes,after which time they were fixed and processed for immuno-electronmicroscopy. FIG. 1 shows the cell prior to 37° C. incubation. J591 canbe seen bound to the cell along the external aspect of the cellmembrane. In this Figure, “M” denotes the cell's mitochondria, and “N”denotes its nucleus. FIG. 2 shows the cell after incubation at 37° C.for 5 minutes. The arrow indicates formation of a clathrin-coated pit.In FIG. 3, which shows the cell after a 10 minute 37° C. incubation,pinching off or endocytosis of the clathrin-coated pit can be seen, asindicated by the arrow. FIG. 4 shows that, after incubation at 37° C.for 15 minutes, monoclonal antibody J591 is contained in endocyticvesicles within the cell, as indicated by the arrows. As can be seen inFIG. 5, after incubation at 37° C. for 15 minutes, monoclonal antibodyJ591 is also contained within endosomes, as indicated by the arrows.

Example 12 Sequencing of the Variable Region of Monoclonal Antibody J591

Total RNA was prepared from 10⁷ murine hybridoma J591 cells. A sample ofthe conditioned medium from these cells was tested for binding to thespecific antigen for J591 on prostate cells. The conditioned medium waspositive by both ELISA and Western Blot for binding to the antigen.

VH and VK cDNA were prepared using reverse transcriptase and mouse κconstant region and mouse IgG constant region primers. The first strandcDNAs were amplified by PCR using a variety of mouse signal sequenceprimers (6 for VH and 7 for VK). The amplified DNAs were gel-purifiedand cloned into the vector pT7Blue.

The VH and VK clones obtained were screened for correct inserts by PCR,and the DNA sequence of selected clones was determined by the dideoxychain termination method.

Excluding the primer region (as the sequence of this depended on thesequence of the primer that was used), all the VH clones obtained gaveidentical sequence. This sequence was obtained from clones produced withthree different 5′ primers. One clone had one base pair change withinthe signal sequence, and one clone contained an aberrant PCR product.Using the sequencing strategy shown in FIG. 6, the nucleotide sequencefor the heavy chain was obtained. It is designated SEQ. ID. No. 1 and ispresented in FIG. 7, along with the nucleotide sequence of thecorresponding reverse, non-coding strand (designated SEQ. ID. No. 2).These sequences include part of the signal sequence and part of theconstant region of the antibody. The corresponding deduced amino acidsequences of J591 VH, designated SEQ. ID. No. 3, SEQ. ID. No. 4, andSEQ. ID. No. 5, are also shown in FIG. 7. The coding strand of the J591heavy chain's variable region (exclusive of signal sequence and constantregion components) has the following nucleotide sequence (designatedSEQ. ID. No. 6): GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAGGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCATACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAACATCAATCCTAACAATGGTGGTACCACCTACAATCAGAAGTTCGAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTAACATCTGAGGATTCTGCAGTCTATTATTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

The reverse, non-coding strand of the J591 heavy chain's variable region(exclusive of signal sequence and constant region components) has thefollowing nucleotide sequence (designated SEQ. ID. No. 7):TGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCAAAGTTCCAACCAGCTGCACAATAATAGACTGCAGAATCCTCAGATGTTAGGCTGCGGAGCTCCATGTAGGCTGTACTGGAGGACTTGTCTACAGTCAATGTGGCCTTGTCCTCGAACTTCTGATTGTAGGTGGTACCACCATTGTTAGGATTGATGTTTCCAATCCACTCAAGGCTCTTTCCATGGCTCTGCTTCACCCAGTGTATGGTATATTCAGTGAATGTGTATCCAGAAGTCTTGCAGGATATCCTCACTGAAGTCCCAGGCTTCACCAGTTCAGGTCCAGACTGTTGCAGCTGGACCTC

The protein sequence corresponding to the J591 heavy chain's variableregion (exclusive of signal sequence and constant region components) hasthe following nucleotide sequence (designated SEQ. ID. No. 8):EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGW NFDYWGQGTTLTVSS

The J591 VH is in Mouse Heavy Chains Subgroup IIA (Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services (1991) (“Kabat”), which is hereby incorporatedby reference). The sequence of J591 VH is compared to the consensussequence for this subgroup in FIG. 8.

In contrast to the VH, more than one VK sequence was obtained. Out ofthe 15 VK clones examined, four gave the sequence of an aberrant mouseIgκ from the fusion partner (Carol et al., Molecular Immunology,25:991-995 (1988), which is hereby incorporated by reference). Theseclones originated from two specific 5′ primers. No further work was donewith these clones of the remaining clones, ten gave identical nucleotidesequences, and one clone, VK17, gave an alternative VK sequence. The tenidentical clones originated from three 5′ primers (different from thetwo that gave the aberrant sequence), one of which also produced VK17.The sequencing strategy that was employed is shown in FIG. 9.

The nucleic acid sequence of J591 VK corresponding to the ten identicalclones (designated SEQ. ID. No. 9) is presented in FIG. 10, along withthe nucleic acid sequence of the corresponding reverse, non-codingstrand (designated SEQ. ID. No. 10) and the deduced amino acidsequences, which are designated SEQ. ID. No. 11, SEQ. ID. No. 12, andSEQ. ID. No. 13. These sequences include part of the signal sequence andpart of the constant region of the antibody. The coding strand of theJ591 light (kappa) chain's variable region (exclusive of signal sequenceand constant region components) corresponding to the ten identicalclones has the following nucleotide sequence (designated SEQ. ID. No.14): AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA

The reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to the ten identical clones has the followingnucleotide sequence (designated SEQ. ID. No. 15): .TTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGATAGCTGTAACCCTGTCCACAGTGATAATCTGCAAGGTCTTCAGCCTGCACACTGCTGATGGTCAGAGTGAAATCTGTTGCAGATCCACTGCCTGTGAAGCGATCGGGGACCCCAGTGTACCGGTTGGATGCCCCGTATATCAGCAGTTTAGGAGACTGCTCTGGTTTCTGTTGATACCAGGAAACATAAGTAACCACATTCTCACTGGCCTTGCAGGTCAAGGTGACCCTCTCTCCTACTGACATGGACATGGATTTGGG AGATTGGGTCATTACAATGTT

The protein sequence corresponding to the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to the ten identical clones has the followingnucleotide sequence (designated SEQ. ID. No. 16):NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGG GTKLEIK

The coding strand of the J591 light (kappa) chain's variable region(exclusive of signal sequence and constant region components)corresponding to clone VK17 has the following nucleotide sequence(designated SEQ. ID. No. 17):GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAATGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCT GGGACCATGCTGGACCTGAAA

The reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to clone VK17 has the following nucleotidesequence (designated SEQ. ID. No. 18):TTTCAGGTCCAGCATGGTCCCAGCACCGAACGTGAGAGGATAGCTGTTATATTGCTGACAGAAATAATCTGCCAAGTCTTCAGACTGAACATTAGTAATGGTGAGAGTGAAGTCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACTCCAGTGTGCCGAGTGGATGCCCAATAAATCAGTAGTTTAGGAGATTGTCCTGGTTTCTGTTGATACCAGTCTACAGCAGTACCCACATCTTGACTGGCCTTACAGATGATGCTGACCCTGTCTCCTACTGATGTGGACATGAATTTGTG AGACTGGGTCATCACAATGTC

The protein sequence corresponding to the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to clone VK17 has the following nucleotidesequence (designated SEQ. ID. No. 19):DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGA GTMLDLK

J591 VK is in the Mouse Kappa Chains Subgroup V (Kabat, which is herebyincorporated by reference). The sequence of J591 VK corresponding to theten identical clones is compared to the consensus sequence for thesubgroup in FIG. 11.

Preferred J591's are those having heavy chain variable region DNA codingstrand sequences corresponding to SEQ. ID. No. 6 and non-coding strand(reverse) sequences corresponding to SEQ. ID. No. 7. The heavy chainvariable region of J591 preferably has an amino acid sequencecorresponding to SEQ. ID. No. 8. The light chain variable region of J591preferably has a DNA coding strand sequence corresponding to SEQ. ID.No. 17, a DNA non-coding strand (reverse) sequence corresponding to SEQ.ID. No. 18, and a amino acid sequence corresponding to SEQ. ID. No. 19.

Example 13 Immunohistochemical Staining of Normal and Cancer Tissues

Cancer tissues from 23 carcinomas were pre-cooled in liquid nitrogen,snap-frozen in OCT compound (Miles, Elkhart, Ind.) on dry ice, andstored at −80° C. Cryostat tissue sections (5 μm) were fixed in coldacetone (4° C.) for 10 minutes. mAbs (5 μg/ml or hybridoma supernatants)were incubated for 1 hour at room temperature. Antibody binding wasdetected using rabbit anti-mouse Ig-peroxidase (Dako, Carpinteria,Calif.) as a secondary antibody and DAB (Sigma, St. Louis, Mo.) aschromogen. Isotype-matched irrelevant antibody was used as negativecontrol.

mAbs J591, J533, J415, and E99 reacted strongly with vascular endotheliain all 23 carcinomas studied, including 9/9 renal, 5/5 urothelial, 6/6colon, 1/1 lung, and 1/1 breast carcinomas, and 1/1 metastaticadenocarcinoma to the liver. FIGS. 2A-2F, respectively, show theimmunohistochemical reactivity of mAb J591 to neovasculature of renal,urothelial, colon, lung, and breast carcinomas, and metastaticadenocarcinoma to the liver.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

1. A method of ablating or killing cancerous cells comprising: providinga biological agent which, when contacted with an extracellular domain ofprostate specific membrane antigen, binds to the extracellular domain ofprostate specific membrane antigen and contacting vascular endothelialcells proximate to the cancerous cells with the biological agent underconditions effective to permit both binding of the biological agent tothe vascular endothelial cells proximate to the cancerous cells andablating or killing of the cancerous cells.
 2. A method according toclaim 1, wherein the biological agent kills or ablates the vascularendothelial cells proximate to the cancerous cells, thereby killing orablating the cancerous cells by reducing blood flow thereto.
 3. A methodaccording to claim 1, wherein the cancerous cells are renal cancerouscells, urothelial cancerous cells, colon cancerous cells, rectalcancerous cells, lung cancerous cells, breast cancerous cells, orcancerous cells of metastatic adenocarcinoma to the liver.
 4. A methodaccording to claim 1, wherein the biological agent is an antibody orbinding portion thereof, probe, or ligand.
 5. A method according toclaim 1, wherein the biological agent, when contacted with anextracellular domain of prostate specific membrane antigen, isinternalized with the prostate specific membrane antigen.
 6. A methodaccording to claim 1, wherein said contacting is carried out in a livingmammal and comprises: administering the biological agent to the mammalunder conditions effective to permit both binding of the biologicalagent to vascular endothelial cells proximate to the cancerous cells andkilling of the cancerous cells.
 7. A method according to claim 6,wherein said administering is carried out orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes.
 8. A method according to claim 4, wherein an antibodyis used in carrying out said method, the antibody being selected fromthe group consisting of a monoclonal antibody and a polyclonal antibody.9. A method according to claim 8, wherein the antibody is selected fromthe group consisting of an E99, a J415, a J533, and a J591 monoclonalantibody.
 10. A method according to claim 8, wherein the antibody is amonoclonal antibody produced by a hybridoma cell line having an ATCCAccession Number selected from the group consisting of HB-12101,HB-12109, HB-12127, and HB-12126.
 11. A method according to claim 4,wherein a binding portion of an antibody is used in carrying out saidmethod, the binding portion being selected from the group consisting ofan Fab fragment, an F(ab′)₂ fragment, and an Fv fragment.
 12. A methodaccording to claim 4, wherein the probe or ligand is used in carryingout said method.
 13. A method according to claim 1, wherein thebiological agent is bound to a substance effective to kill or ablate thecancerous cells upon binding of the biological agent to vascularendothelial cells proximate to the cancerous cells.
 14. A methodaccording to claim 13, wherein the substance effective to kill or ablatethe cancerous cells is a cytotoxic drug.
 15. A method according to claim14, wherein the cytotoxic drug is selected from the group consisting oftherapeutic drug, a compound emitting radiation, molecules of plant,fungal, or bacterial origin, biological proteins, and mixtures thereof.16. A method according to claim 4, wherein the antibody is effective toinitiate an endogenous host immune function.
 17. A method according toclaim 16, wherein the endogenous host immune function iscomplement-mediated cellular cytoxicity.
 18. A method according to claim16, wherein the endogenous host immune function is antibody-dependentcellular cytoxicity.
 19. A method according to claim 1, wherein thebiological agent is in a composition further comprising aphysiologically acceptable carrier, excipient, or stabilizer.
 20. Amethod according to claim 1, wherein the biological agent is in acomposition further comprising a pharmaceutically acceptable carrier,excipient, or stabilizer.
 21. A method of detecting cancerous tissue ina biological sample comprising: providing an biological agent which,when contacted with an extracellular domain of prostate specificmembrane antigen, binds to the extracellular domain of prostate specificmembrane antigen, wherein the biological agent is bound to a labeleffective to permit detection of vascular endothelial cells proximate toor within the cancerous tissue upon binding of the biological agent tothe vascular endothelial cells proximate to or within the canceroustissue; contacting the biological sample with the biological agenthaving a label under conditions effective to permit binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue in the biological sample; and detecting apresence of any cancerous tissue in the biological sample by detectingthe label.
 22. A method according to claim 21, wherein the canceroustissue is renal cancerous tissue, urothelial cancerous tissue, coloncancerous tissue, rectal cancerous tissue, lung cancerous tissue, breastcancerous tissue, or cancerous tissue of metastatic adenocarcinoma tothe liver.
 23. A method according to claim 21, wherein the biologicalagent is an antibody or binding portion thereof, probe, or ligand.
 24. Amethod according to claim 21, wherein the biological agent, whencontacted with an extracellular domain of prostate specific membraneantigen, is internalized with the prostate specific membrane antigen.25. A method according to claim 21, wherein said contacting is carriedout in a living mammal and comprises: administering the biological agentto the mammal under conditions effective to permit binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue in the biological sample.
 26. A methodaccording to claim 25, wherein the label is a short-range radiationemitter.
 27. A method according to claim 25, wherein said administeringis carried out orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intraversal instillation, byintracavitary or intravesical instillation, intraocularly,intraarterially, intralesionally, or by application to mucous membranes.28. A method according to claim 23, wherein an antibody is used incarrying out said method, said antibody being selected from the groupconsisting of a monoclonal antibody and a polyclonal antibody.
 29. Amethod according to claim 28, wherein the antibody is selected from thegroup consisting of an E99, a J415, a J533, and a J591 monoclonalantibody.
 30. A method according to claim 28, wherein the antibody is amonoclonal antibody produced by a hybridoma cell line having an ATCCAccession Number selected from the group consisting of HB-12101,HB-12109, HB-12127, and HB-12126.
 31. A method according to claim 23,wherein a binding portion of an antibody is used in carrying out saidmethod, the binding portion being selected from the group consisting ofan Fab fragment, an F(ab′)₂ fragment, and an Fv fragment.
 32. A methodaccording to claim 23, wherein a probe or ligand is used in carrying outsaid method.
 33. A method according to claim 21, wherein the label isselected from the group consisting of a fluorescent label, a radioactivelabel, a nuclear magnetic resonance active label, a luminescent label,and a chromophore label.
 34. A method according to claim 21, wherein thebiological agent is in a composition further comprising aphysiologically acceptable carrier, excipient, or stabilizer.
 35. Amethod according to claim 21, wherein the biological agent is in acomposition further comprising a pharmaceutically acceptable carrier,excipient, or stabilizer.
 36. A method according to claim 21, whereinsaid contacting is carried out in a sample of serum or urine.
 37. Amethod according to claim 21, wherein said contacting is carried out ina tissue biopsy sample.